The Diabetes Control and Complications Trial (DCCT) established that the microvascular complications of diabetes can be prevented by maintaining near normal glycemic control in patients with type 1 diabetes (T1D). This seminal outcome has provided a strong impetus for developing effective tolerogenic strategies for a cell replacement via pancreas or isolated islet transplantation in T1D patients. The recent success of the "Edmonton protocol" was a major advance in the field of islet transplantation. However, despite initial achievement of an insulin independent state, a progressive loss of beta cell function culminates in the recurrence of diabetes in a majority of these recipients. Thus, the Edmonton approach, which targets the T lymphocyte compartment alone, seems insufficient in preventing immunological rejection and recurrent anti-beta cell autoimmunity. Despite the critical role of T lymphocytes in these two processes, it is established that a concomitant and specific B lymphocyte response against beta-cell derived allo- and auto-antigenic epitopes also occurs. In rodent studies we demonstrated that interruption of B lymphocyte function curtails the T cell mediated destruction of islet allografts. Thus, we hypothesize that an induction immunotherapy regimen which targets both the B- and T lymphocyte compartments, will promote immunological tolerance to islet allografts. Our preclinical studies test this premise in non-human primates (NHPs) and indicate that the combined use of anti-B and T-lymphocyte antibodies (i.e., Rituximab and Thymoglobulin) results in long-term islet allograft survival without the need for maintenance therapy with a calcineurin inhibitor (CNI) agent. In the present application we propose to determine the efficacy of combined B- and T- lymphocyte directed immunotherapy for promoting immunological tolerance to islet allografts in T1D patients. We will initiate a clinical trial which will: 1) build upon our experience with the "Edmonton protocol", by incorporating the B lymphocyte specific monoclonal antibody, Rituximab, into its induction regimen and 2) assess the efficacy of a combined induction regimen including Thymoglobulin and Rituximab followed by CNI-free maintenance monotherapy with Rapamycin. Importantly, the latter protocol, which parallels that used in our preclinical NHP studies, will permit the inclusion of T1D patients with microalbuminuria into islet transplantation trials. A series of prospective in vivo metabolic studies will be undertaken to specifically evaluate beta cell function and secretory capacity following transplantation that 1) includes Rituximab and 2) eliminates CNI agents. Our mechanistic studies are designed to test the hypothesis that Rituximab immunotherapy provides a "tolerogenic window" for the reconstituting B lymphocyte repertoire, during which allo- and auto-reactive clones with a transitional phenotype are subject to negative selection. Following islet cell transplantation we will: 1) monitor the development of alloantibodies to HLA antigens and autoantibodies to islet antigens, 2) analyze the immunoglobulin repertoire for clonal persistence and heterogeneity using antibody CDR3 spectratyping, 3) survey the cytokine profiles of autoantigen-specfic T cells reactive to islet beta-cells by ELISpot, and 4) perform immunophenotyping and functional assays of circulating lymphocytes to assess global alterations in lymphocyte differentiation and energy. Overall, the proposed studies will determine whether a balanced immunotherapy regimen targeting the B- and T- lymphocyte compartments promotes a state of immunological tolerance to islet allografts, while obviating the need for chronic immunosuppression.